Escobar L, Salvador C, Contreras M, Escamilla J E
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México.
Anal Biochem. 1990 Jan;184(1):139-44. doi: 10.1016/0003-2697(90)90026-6.
A method for recording O2 concentrations in nonconducting organic media with the Clark oxygen electrode was developed. Spontaneous oxidation of Na2S2O4 and the enzymatic reduction of NaBO3 or H2O2 by bovine liver catalase trapped in hydrated micelles of dioctylsulfosuccinate (AOT)/toluene were used as model systems. O2 titration with the above systems showed that air-saturated 1.6 M H2O/0.2 M AOT/toluene media contain seven times more O2 (1.4 mM) than aqueous solutions (0.2 mM). The measured Km values of catalase for NaBO3 and H2O2 in organic media were Kmov = 15 and 17 mM, respectively, whereas in aqueous buffer the values were 45 and 54 mM. In the toluene media, catalase activity increased with the W0 (H2O/AOT molar ratio) of the micellar preparation, reaching maximal activity at W0 = 10-12; under this condition, the catalytic center activity (Kp) of H2O2 was 7 x 10(6) min-1, similar to that obtained in the aqueous buffer (H2O2 = 7 x 10(6) min-1). It was found that the optimal pH for catalase in toluene media (pH 8.0) was shifted 1.0 unit compared to that in the aqueous buffer (pH 7.0). On the other hand, catalase was severely inhibited by NaN3 in both media. Thus, polarography based on the Clark oxygen electrode seems to be an easy, rapid, and sensitive technique for studying enzyme reactions consuming or evolving O2 in apolar media.
开发了一种使用克拉克氧电极记录非导电有机介质中氧气浓度的方法。以连二亚硫酸钠的自发氧化以及被困在二辛基磺基琥珀酸钠（AOT）/甲苯水合胶束中的牛肝过氧化氢酶对硼酸钠或过氧化氢的酶促还原作为模型系统。用上述系统进行的氧气滴定表明，空气饱和的1.6 M水/0.2 M AOT/甲苯介质中的氧气含量（1.4 mM）是水溶液（0.2 mM）的7倍。在有机介质中测得的过氧化氢酶对硼酸钠和过氧化氢的Km值分别为Kmov = 15 mM和17 mM，而在水性缓冲液中的值分别为45 mM和54 mM。在甲苯介质中，过氧化氢酶活性随胶束制剂的W0（水/AOT摩尔比）增加，在W0 = 10 - 12时达到最大活性；在此条件下，过氧化氢的催化中心活性（Kp）为7×10⁶ min⁻¹，与在水性缓冲液中获得的值（过氧化氢 = 7×10⁶ min⁻¹）相似。发现甲苯介质中过氧化氢酶的最佳pH（pH 8.0）与水性缓冲液中的最佳pH（pH 7.0）相比偏移了1.0个单位。另一方面，在两种介质中叠氮化钠都能严重抑制过氧化氢酶。因此，基于克拉克氧电极的极谱法似乎是一种用于研究在非极性介质中消耗或释放氧气的酶反应的简便、快速且灵敏的技术。
